Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 September 2018 doi:10.20944/preprints201809.0299.v1 Peer-reviewed version available at Regional Studies in Marine Science 2020, 35; doi:10.1016/j.rsma.2020.101109 Molecular approach to the identification and phylogenetic biogeography of snail Telescopium telescopium using mt-COI gene sequences Satheesh Kumar Palanisamy1,2 , Prasanna Kumar Chinnamani3, Purushothaman Paramasivam4, Umamaheswari Sundaresan5 1Central Marine Fisheries Research Institute, Kochi - 682018, India 2Department of Zoology, School of Natural Science and Ryan Institute, National University of Ireland Galway (NUI Galway), University Road, H91TK33, Galway 3Institute of Marine Microbes and Ecosphere, State Key Laboratory for Marine Environmental Sciences, Xiamen University, Xiamen, Fujian, (361102) China PR. 4Crustacean Fisheries Division, Central Marine Fisheries Research Institute, Kochi - 682018, India. 5Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India Correspondence: [email protected] Tel.: +353899608763 Abstract: We aimed to apply DNA barcoding tool for the molecular identification of horn snails T. telescopium using mitochondrial cytochrome oxidase I gene (mt-COI) and to investigate their evolutionary relationship along with location-specific bio-geographical variations. The molecular data sets of this study indicate that strong probability of T. telescopium species taxonomic confirmation using mt-COI sequences. Results of the phylogenetic analysis suggest that Telescopium sp. was monophyletic with disseminated nodes and the evolution of group II originated from group I. The substantial genetic distance among the mt-COI sequences (0.005 to 0.184) were noticed. Large divergence between the south-west coast of India and Australia region population indicates limited gene flow between the two continents. Our study suggests that the genera Telescopium is globally ubiquitous but genetically showing inter-region differentiation. We conclude that mt-COI gene can be used to identify gastropod T. telescopium species. Keywords: Barcoding, biodiversity, genetics, mollusc, phylogeny, snails, taxonomy 1 © 2018 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 September 2018 doi:10.20944/preprints201809.0299.v1 Peer-reviewed version available at Regional Studies in Marine Science 2020, 35; doi:10.1016/j.rsma.2020.101109 1. Introduction Indian coastal waters are receiving more attention from the scientific community due to their enormous fishery and shellfish potential resources [1]. In the Indian Ocean, Bay of Bengal waters hosts an extent range of molluscan (bivalves and gastropods) faunal distribution, species diversity and they are ecologically important to many communities [2]. However, this group diversity is highly underestimated [3]. Molluscs are the most diverse marine phylum and represent higher species diversity has resulted in a considerable taxonomic problem [4]. Some species in Indian coast remain uncertain; there is a need to incorporate molecular methods. Molecular markers especially mitochondrial DNA cytochrome oxidase I (mt-COI) are considered as a better choice for species discrimination due to non-recombinant and environment independent nature [4]. The application of DNA barcoding brings substantial benefits to other fields, such as border biosecurity [5], biodiversity conservation [6], ecological monitoring [7]. DNA barcoding has successfully utilized for the taxonomic confirmation of marine organisms, the molecular phylogeny of gastropod molluscs is extensively studied using morphometric characteristics and DNA sequences [8]. The standard pairwise distance method such as Neighbour-Joining tree (NJ) based on Kimura 2-parameter distance (K2P) and GC content divergence assessment is currently the principal approach used to analyze patterns of diversity with COI barcode region. It has been informative at the species-level discrimination across the range of several groups from terrestrial, freshwater and marine environment [9]. The accuracy of such results depends particularly on the delineation between intra-specific variation and inter-specific DNA sequence divergence [10]. The strong molecular identification of marine gastropods requires the creation of comprehensive reference library of DNA barcode matching of each specimen to a diagnostic barcode sequence [11]. Gastropod mollusc Telescopium telescopium lives in mud-flats of mangrove forest in throughout the coastlines of Indian Ocean waters. Mangrove horn snail or mud whelks T. telescopium is the only living organism of this genus from the family Potamidae (Gastropoda), found abundantly and frequently dominated in mud-flats of mangrove forest in West and Central Indo-Pacific regions due in part of its central locations at the meeting of oceanic waters has the greatest diversity of mangrove forest. The distribution of this species is likely impacted locally by the loss of habitat [e.g. mangrove deforestation, marine pollution] [12]. So far, no reliable information available to deal the estimated population of this species in Indian waters. Houbrick et al. [13] was reported the revised systematics and phylogeny of this taxa, following that Bandel et al. [14] was recognized two sub families, Potamidinae and Telescopinae, the 2 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 September 2018 doi:10.20944/preprints201809.0299.v1 Peer-reviewed version available at Regional Studies in Marine Science 2020, 35; doi:10.1016/j.rsma.2020.101109 later on included all the living potamidids except the genus Potamides in the family Potamididae. With the recent development of molecular techniques, the evolutionary relationship of Potamididae and their ecological association with mangrove ecosystem has also been studied using mt-COI sequence data [15]. The further research of this genus will reveal to complete understanding of the evolutionary relationship of this species in family Potamidae, ecological association with mangrove forest and their potential application in pollution monitoring [16] and marine bio-discovery program [17, 18]. The DNA sequence of mt-COI gene data facilitate not only the inference the phylogenetic relationships of fauna but also providing an efficient method for species-level identification under the term of molecular taxonomy or DNA Barcoding [19]. 1.1 Evolutionary significant of horn snail Mangrove horn snail or mud whelks T. telescopium is the only living organism of this genus from the family Potamidae (Gastropoda), found abundantly and frequently dominated in mud-flats of mangrove forest in West and Central Indo-Pacific regions due in part of its central locations at the meeting of oceanic waters has the greatest diversity of mangrove forest. The distribution of this species is likely impacted locally by the loss of habitat [e.g. mangrove deforestation, marine pollution] [12]. So far, no reliable information available to deal the estimated population of this species in Indian waters. Houbrick et al. [13] was reported the revised systematics and phylogeny of this taxa, following that Bandel et al. [14] was recognized two sub families, Potamidinae and Telescopinae, the later on included all the living potamidids except the genus Potamides in the family Potamididae. With the recent development of molecular techniques, the evolutionary relationship of Potamididae and their ecological association with mangrove ecosystem has also been studied using mt-COI sequence data [15]. The further research of this genus will reveal to complete understanding of the evolutionary relationship of this species in family Potamidae, ecological association with mangrove forest and their potential application in pollution monitoring [16] and marine bio-discovery program [17, 18]. The DNA sequence of mt-COI gene data facilitate not only the inference the phylogenetic relationships of fauna but also providing an efficient method for species-level identification under the term of molecular taxonomy or DNA Barcoding [19]. The aim of this present study, to improve the systematics of the genus Telescopium by the integration of morphometric characteristics and DNA barcoding of T. telescopium shells found in the Bay of Bengal waters. ii) to determine their evolutionary relationship by bio- geographical variations based on mt-COI sequences. 3 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 September 2018 doi:10.20944/preprints201809.0299.v1 Peer-reviewed version available at Regional Studies in Marine Science 2020, 35; doi:10.1016/j.rsma.2020.101109 2. Material and methods 2.1 Collection of samples Horn snails used in this study were collected in Parangipettai coast, Bay of Bengal - India during March to August 2011 and their taxonomy was identified using [13]. All the specimens (n=10) were preserved in 95% ethanol for DNA molecular analysis. 2.2 DNA Isolation and Sequencing Total DNA was extracted from the muscle tissue/eggs of each specimen using Qiagen DNA Easy blood and tissue kit (Hilden, Germany). Universal Primers LCO 1490 and HCO2198 as described in Folmer et al. [20] were used for the amplification of a fragment of mt-COI gene. PCRs were performed in a 12.5 µl volume with 6.25 µl of 10% trehalose, 1.25 µl PCR buffer [200 mM Tris– HCl (pH 8.4), 500 mM KCl], 2 .85 µl of nuclease free water, 0.625 µl MgCl2 (50 mM), 0.5 µl of each primer (10 µM), and 1 µl of DNA template.